作者: Stalmans, Ingeborg ; Vandewalle, Evelien ; Bergen, Tine Van ; Moons, Lieve ; van Pelt, Jos ; Sergeys, Jurgen ; Lefevere, Evy ; Castermans, Karolien ; Hollanders, Karolien ; Kindt, Nele ; Hove, Inge Van

PURPOSEDiabetic retinopathy (DR) is characterized by an early stage of inflammation and vessel leakage, and an advanced vasoproliferative stage. Also, neurodegeneration might play an important role in disease pathogenesis. The aim of this study was to investigate the effect of the Rho kinase (ROCK) inhibitor, AMA0428, on these processes.METHODSThe response to ROCK inhibition by AMA0428 (1 µg) was studied in vivo using the murine model for streptozotocin (STZ)-induced diabetes, focusing on early non-proliferative DR features and the oxygen-induced retinopathy (OIR) model to investigate proliferative DR. Intravitreal (IVT) administration of AMA0428 was compared with murine anti-VEGF-R2 antibody (DC101, 6.2 µg) and placebo (H₂O/PEG; 1C8). Outcome was assessed by analyzing leukostasis using fluorescein isothiocyanate coupled concanavalin A (FITC-ConA) and vessel leakage (bovine serum albumin conjugated with fluorescein isothiocyanate; FITC-BSA)/neovascularization and neurodegeneration by immunohistological approaches (hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL), Brn3a). ELISA and Western blotting were employed to unravel the consequences of ROCK inhibition (1 µM AMA0428) on myosin phosphatase target protein (MYPT)-1 phosphorylation, endothelial nitric oxide synthase (eNOS) phosphorylation, and vascular endothelial growth factor (VEGF) levels in retinas of diabetic mice, on NF-κβ activity and ICAM-1 expression in endothelial cells (ECs).RESULTSIn vivo, AMA0428 significantly reduced vessel leakage and neovascularization, respectively, in the STZ and OIR model, comparable to DC101 therapy. Additionally, the ROCK inhibitor decreased neurodegeneration in both models and inhibited leukostasis by 30% (p < 0.05) in the STZ model (p < 0.05), while DC101 had no positive effect on the outcome of these latter processes. ROCK activity was upregulated in the diabetic retina and AMA0428 administration resulted in decreased phospho-MYPT-1, enhanced phospho-eNOS, and reduced VEGF levels. In vitro, AMA0428 interfered with NF-κβ activity, thereby inhibiting ICAM-1 expression in ECs.CONCLUSIONSTargeting ROCK with AMA0428 effectively attenuated outcome in an early DR model (STZ) and a late vasoproliferative retinopathy model (OIR). These findings make AMA0428 a promising candidate with an additional anti-inflammatory and neuroprotective benefit for DR patients, as compared with anti-VEGF treatment.

2015-02-27·Investigative Ophthalmology & Visual Science2区 · 医学

The Effect of AMA0428, a Novel and Potent ROCK Inhibitor, in a Model of Neovascular Age-Related Macular Degeneration

2区 · 医学

ArticleOA

作者: Moons, Lieve ; Kindt, Nele ; Van Bergen, Tine ; Castermans, Karolien ; Vandewalle, Evelien ; Hollanders, Karolien ; Leysen, Dirk ; Stalmans, Ingeborg

PURPOSERho kinase (ROCK) is associated with VEGF-driven angiogenesis, as well as with inflammation and fibrosis. Therefore, the effect of AMA0428, a novel ROCK inhibitor, was studied in these processes, which highly contribute to the pathogenesis of neovascular AMD.METHODSThe effect of AMA0428 (0.5-5.0 μM) on human umbilical vein endothelial cells (HUVECs), human brain microvascular endothelial cells (HBMECs), and human brain microvascular pericytes (HBVPs) was determined using cell viability (WST-1), apoptosis (caspase 3/7), and migration (scratch and under-agarose) assays. The in vivo response was investigated using a laser-induced choroidal neovascularization (CNV) mouse model, in which intravitreal injections of AMA0428, murine anti-VEGF-R2 mAb (DC101), or placebo was given. Outcome was assessed by analysis of inflammation (CD45), angiogenesis (FITC-dextran), vessel leakage (Texas Red-conjugated Dextran and FITC-labeled lectin) and fibrosis (Sirius Red/Collagen I).RESULTSThe AMA0428 dose-dependently reduced proliferation and VEGF-induced migration of HUVEC and HBMEC (P < 0.05). No significant effect was seen on HBVP proliferation; however, migration and pericyte recruitment were enhanced (P < 0.05) by AMA0428 administration. There was no apoptosis induction. The AMA0428 significantly reduced CNV and vessel leakage 2 weeks after laser treatment, comparable to DC101. In addition, AMA0428 inhibited inflammation on day 5 by 42% (P < 0.05) and collagen deposition on day 30 by 43% (P < 0. 05), whereas DC101 had no effect on inflammation nor on fibrosis.CONCLUSIONSThe results suggest that targeting ROCK with AMA0428 not only reduces neoangiogenesis, but also blocks inflammation and fibrosis (contrary to VEGF suppression). These results point to a potential therapeutic benefit of ROCK inhibition in neovascular AMD.

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